1. Field of the Invention
The present invention relates generally to microfluidic devices for mixing suspensions of affinity capture beads in fluidized biological samples, and for apparatuses and methods for ligand capture, depletion, preconcentration, washing, and labeling, followed by analysis, by means of affinity capture beads.
2. Description of the Related Arts
Biological analytes of relevance to clinical, biological, or environmental testing frequently are found at low concentrations in complex fluid mixtures. It is important to capture, concentrate, and enrich the specific analyte away from background inhibitory or interfering matrix components that can limit the sensitivity and/or specificity of analyte detection assays. Specific analytes include but are not limited to nucleic acids, proteins, including for example antigens or antibodies, prokaryotic or eukaryotic cells, and viruses, and small molecules such as drugs and metabolites. Conventional sample preparation methods include centrifugation, solid phase capture, selective precipitation, filtration, and extraction. These methods are not generally amenable to efficient automation and integration with subsequent assay steps, especially in a manner compatible with the development of point of care testing.
Another proven method for preparation of samples takes advantage of magnet fields to capture magnetic particles that have been derivatized to bind a ligand that is linked to the analyte of interest (i.e., capture), or a ligand that is linked to an inhibitor or interference (i.e., depletion). Both methods have been used to prepare the analyte of interest for detection.
Magnetic particles have found a number of uses in biomedical research and diagnostics. A seminal procedure for magnetic capture of cells, bacteria, and viruses is disclosed in U.S. Pat. Nos. 3,970,518 and 4,018,886. The method according to this invention for sorting out and separating a select cell population from a mixed cell population comprises the steps of applying to the surface of small magnetic particles a coating of an antibody to the select cell, bacteria or virus population: moving these antibody-coated magnetic particles through a liquid containing the mixed population whereby the members of the cell, bacteria or virus population become affixed to the antibody coatings on the particles, and separating the coated magnetic beads with such members attached thereto from the rest of the mixed population. These methods also included a “cleaving” step for releasing the bound members. Mixing is provided by an impeller mounted in a modified funnel. Typical incubation times were said to be less than an hour to as much as a day, the lower the concentration, the longer the incubation required. Magnetic particles were said to include ferrites, perovskites, chromites, or magnetoplumbites, and more generally, ferromagnetic, ferrimagnetic or superparamagnetic particles, although it is obvious that permanently magnetic particles will readily clump whereas superparamagnetic particles remain monodisperse until a magnetic field is applied. Hersh, in U.S. Pat. No. 3,933,997 described the use of magnets to capture digoxin from solution.
Other patents describing analytical applications for magnetic particles include U.S. Pat. Nos. 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Recent literature citations include: Shaikh K A et al. 2005. A modular microfluidic architecture for integrated biochemical analysis. PNAS 102; 9745-9750; Fuentes M et al. 2006. Detecting minimal traces of DNA using DNA covalently attached to superparamagnetic nanoparticles and direct PCR-ELISA. Biosensors Bioelectronics 21:1574-1580, and Sirigireddy K R and Ganta R R. 2005. Multiplex detection of Ehrlichia and Anaplasma species pathogens in peripheral blood by real-time reverse-transcriptase-polymerase chain reaction. J Mol Diagnostics 7:308-316.
Magnetic beads can be classified on the basis of size as large (1.5 to about 50 microns), small (0.7-1.5 microns), or colloidal (<200 nm), which are also referred to as nanoparticles. Typical of large magnetic beads (>1.5 microns to about 50 microns) are those described by Ugelstad in U.S. Pat. No. 4,654,267 and manufactured by Dynal® (Oslo, Norway). The Ugelstad process involves the synthesis of polymer beads which are caused to swell and magnetite crystals are embedded in the swelled beads.
Large magnetic beads have been found to be advantageous especially when added to viscous or particulate specimens such as feces or blood since they are readily separated with simple laboratory magnets because of the mass of magnetic material per particle. However, limitations of their use occur since these large particles are not colloidal, and do not diffuse. Consequently, the specimen volume and selectivity of capture requires incubation times and adequate mixing to enable the molecular collision of the analyte with the capture particle. Mixing is also critical to subsequent wash steps. Tube methods exhibit inconsistent and long (15-90 minute) mixing and are not readily automated and integrated within an assay method.
Magnetic beads for affinity capture and bead capture devices are readily available, for example from: Dynal Biotech ASA, Oslo, Norway; BD Biosciences, San Jose, Calif.; and New England Biolabs, Beverly, Mass., among others. The Dynal ClinExVivo MPC® typifies the capture devices, which rely on rocking or rotation for mixing.
Monodisperse, non-colloidal, latex beads and ion exchange beads have also been used in preconcentration steps for detection of biological analytes. Sedimentation fractionation of DNA with derivatized latex particles was demonstrated as early as 1987 (Wolf S F et al. 1987. Rapid hybridization kinetics of DNA attached to submicron latex particles. Nucl Acids Res 15:2911-2926). These particles demonstrated improved capture kinetics of specific targets as compared to planar solid supports and could be concentrated by centrifugation or filtration. Non-specific DNA capture may be achieved with polyethyleneimine or polyvinylpyrrolidone beads, for example.
Microfluidic devices have become popular in recent years for performing analytical testing. Using tools developed by the semiconductor industry to miniaturize electronics, it has become possible to fabricate intricate fluid systems which can be inexpensively mass produced. Systems have been developed to perform a variety of analytical techniques for the acquisition and processing of information.
The ability to perform analyses microfluidically provides substantial advantages of throughput, reagent consumption, and automatability. Another advantage of microfluidic systems is the ability to integrate a plurality of different operations in a single “lap-on-a-chip” device for performing processing of reactants for analysis and/or synthesis. Microfluidic devices may be constructed in a multi-layer laminated structure wherein each layer has channels and structures fabricated from a laminate material to form microscale voids or channels where fluids flow. A microscale or microfluidic channel is conventionally defined as a fluid passage which has at least one internal cross-sectional dimension that is less than 500 μm and typically between about 0.1 μm and about 500 μm.
U.S. Pat. No. 5,716,852, hereby incorporated by reference in its entirety, is an example of a microfluidic device. The '852 patent teaches a microfluidic system for detecting the presence of analyte particles in a sample stream using a laminar flow channel having at least two input channels which provide an indicator stream and a sample stream, where the laminar flow channel has a depth sufficiently small to allow laminar flow of the streams and length sufficient to allow diffusion of particles of the analyte into the indicator stream to form a detection area, and having an outlet out of the channel to form a single mixed stream. This device, which is known as a T-Sensor, allows the movement of different fluidic layers next to each other within a channel without mixing other than by diffusion. A sample stream, such as whole blood, a receptor stream, such as an indicator solution, and a reference stream, which may be a known analyte standard, are introduced into a common microfluidic channel within the T-Sensor, and the streams flow next to each other until they exit the channel. Smaller particles, such as ions or small proteins, diffuse rapidly across the fluid boundaries, whereas larger molecules diffuse more slowly. Large particles, such as blood cells, show no significant diffusion within the time the two flow streams are in contact.
Many different types of valves for use in controlling fluids in microscale devices have been developed. For example, U.S. Pat. No. 6,432,212 describes one-way valves for use in laminated microfluidic structures, U.S. Pat. No. 6,581,899 describes ball bearing valves for use in laminated microfluidic structures, and U.S. patent application Ser. No. 10/114,890, which application is assigned to the assignee of the present invention, describes a pneumatic valve interface, also known as a zero dead volume valve, for use in laminated microfluidic structures. The foregoing patents and patent applications are hereby incorporated by reference in their entirety.
There is general agreement that, in the laminar flow regime characteristic of microfluidic channels, mixing is limited to diffusion. Because of the dimensions involved, wherein diffusional free path lengths are roughly equal the device dimensions, diffusional mixing can be very effective for solutes. This condition enables ribbon flow, T-sensor, and other useful microfluidic phenomena. However, for larger analytes such as cells, bacteria, viral particles, and for macromolecular complexes and linear polymers, diffusional mixing is slow and processes for capture or depletion of these species require prolonged incubation. Diffusional limits on mixing thus present a problem in microfluidic devices where bulk mixing or comminution of a sample and reagents or beads is required. This problem has not been fully solved and methods, devices and apparatuses for improving the mixing arts are being actively sought.